This invention relates to the studies of environmental effects on the health of mammals and, more particularly, to the isolation of mammalian cell mutants that are sensitive to environmental agents.
One area of active research in the environmental field involves the use of mammalian mutant cells that are sensitive to DNA-damaging agents, i.e., environmental agents, e.g., chemical agents, ionizing and ultraviolet radiation, etc. A difficult step in these studies is the initial isolation of a stable, sensitive cell line. In the first instance, only a small number of mutant cells exist in any given cell population. Sensitivity is determined by killing the sensitive cells; yet there must be surviving cells for use in growing a sensitive cell population.
Current technology allows the isolation of only about 500 clones per hour. This means that it takes about 3 months to isolate 10.sup.6 clones, without even considering the additional time needed to observe the clones for responsive to a given environmental agent. The lack of a rapid and reliable method for screening large numbers of mammalian clones for sensitivity to environmental agents has greatly hindered progress in identifying the genes responsible for DNA damage and repair. For instance, there are about ten laboratories actively involved in isolating the genes responsible for resistance to ionizing radiation; all of this work is concentrated in the study of &lt;20 mutant cell lines. The small number of sensitive mutants available has even precluded the determination of the number of complementation groups for repair or radiation-induced DNA damage, a first step in determining the number of genes involved in this complex cellular activity. Current technology also precludes the isolation and testing of enough mutants to study spontaneous mutations, which are thought to occur at 10.sup.-6 or less in a hemizygous cell line.
The development of a method for rapidly isolating mammalian cell mutants sensitive to environmental agents would have significant application to the study of somatic cell genetics, DNA damage repair, toxicity testing, environmental monitoring, and basic cell biology. The ability to isolate large numbers of sensitive mutants could be used to test mutation induction frequencies, while isolation of many mutants sensitive to a given agent would greatly assist in determining complementation patterns. Isolation of new radiation damage repair mutants may allow a description of the mechanisms of DNA damage repair in mammalian cells. The ability to develop mutants sensitive to a variety of harmful environmental agents would allow sensitive biological testing and monitoring procedures. The isolation of mutants sensitive (or resistant) to hormones and growth inhibitory compounds has proven to be a very powerful technique for developing mechanistic descriptions of basic cellular biology.
Thus, a rapid mutant isolation technique would greatly promote the widespread application of mammalian cell mutants to many areas of genetic and biological research. Consequently, it is an object of the present invention to provide a method for the rapid isolation of large numbers of individual cell clones.
Another object of the present invention is to provide a method for the rapid identification of putative sensitive clones after exposure to damaging environmental agents.
Yet another object of the present invention is to provide a method for quickly separating sensitive clones from resistant clones.
One other object of the present invention is to provide a method for the recovery of viable cells from sensitive clones after exposure to a selected test agent.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.